The clinical outcomes of lung cancer remain poor. The targeted delivery of treatment and the implementation of a method to overcome drug resistance are essential for the improvement of cancer therapy. The aim of our study was to assess the treatment effectiveness of engineered extracellular vesicles (EV) carrying both dinaciclib, a potent CDK inhibitor, and the proapoptotic factor TRAIL for a combinatorial lung cancer therapy. We showed that the engineered complexed EV agent, EV-T-Dina, was stable both in vitro and in vivo. Importantly, EV-T-Dina can overcome the drug-resistance of lung cancer cells, and when nebulized and administered by the pulmonary route, it demonstrated high efficacy and satisfactory safety for the treatment of lung cancers. The underlying mechanism for the synergistic killing of cancer cells by dinaciclib and TRAIL was associated with the concomitant downregulation of the anti-apoptotic factors cFLIP, MCL-1, and Survivin. Thus, the aerosolized EV-T-Dina potentially constitutes a novel and effective therapy for lung cancers.The clinical outcomes of lung cancer remain poor, mainly due to the chemoresistance and low bioavailability of systemically delivered drugs. Therefore, novel therapeutic strategies are urgently needed. The TNF-related apoptosis-inducing ligand (TRAIL)-armed extracellular vesicle (EV-T) has proven to be highly synergistic for the killing of cancer cells with the potent cyclin-dependent kinase (CDK) inhibitor Dinaciclib (Dina). However, both optimal drug formulations and delivery strategies are yet to be established to facilitate the clinical application of the combination of EV-T and Dina. We hypothesize that Dina can be encapsulated into EV-T to produce a complexed formulation, designated EV-T-Dina, which can be nebulized for pulmonary delivery to treat lung cancer with potentially improved efficacy and safety. The prepared EV-T-Dina shows good stability both in vitro and in vivo and is very efficient at killing two highly TRAIL-resistant cancer lines. The ability to overcome TRAIL resistance is associated with the concomitant downregulation of the expression of cFLIP, MCL-1, and Survivin by Dina. The EV-T-Dina solution is nebulized for inhalation, showing unique deposition in animal lungs and importantly it demonstrates a significant suppression of the growth of orthotopic A549 tumors without any detectable adverse side events. In conclusion, the aerosolized EV-T-Dina constitutes a novel therapy, which is highly effective and safe for the treatment of lung cancers. As the leading cause of cancer death, lung cancer has the characteristics of high prevalence and frequent metastasis with a very limited efficacy of its first-line treatment. It was estimated that there were 2.2 million new lung cancer cases 11.4%) and 1.8 million deaths (18%) in 2020 worldwide [1.4% and Currently, the prognosis of lung cancer remains poor with an average five-year survival rate of only 16% despite many advances in diagnosis and treatment .The treaChemotherapy remains a primary approach for the treatment of lung cancers , and theThe pulmonary delivery of aerosolized therapeutics may enable topical and more targeted lung cancer medication with maximized dosages and avoid the potential shortcomings of systemic administration . InhalatExtracellular vesicles (EV) are an emerging nanoparticle vector for drug delivery ,13, prodPreviously, we successfully engineered MSCs to produce TNF-related apoptosis-inducing ligand (TRAIL)-armed EVs (EV-T) as an effective anticancer therapy . Recentl2.All cellular culture reagents were purchased from Gibco  unless otherwise stated. Normal human umbilical cord (UC)-derived mesenchymal stem cells (UC-MSCs) and two NSCLC lines A549 and NCI-H23 (abbreviated to H23) were used in this study. A549 cells were luciferase (Luc) transduced. MSCs were purchased from the Shandong Yunzhou Biotechnology Co., Ltd., Zibo, China, and both A549 and H23 were obtained from the Fuheng Biology . A549 and H23 were grown in a DMEM medium containing high sugar (4.5 g/mL) and 10% fetal bovine serum (FBS); MSCs were cultured in the DMEM/F-12 medium supplemented with 10% FBS. Cells were cultured and maintained at 37 °C in a humidified atmosphere containing 5% COg, 10 min at 2000× g, and 30 min at 10,000× g to remove cells and cell debris; and then concentrated by using a centrifugal filter unit . Finally, the cleaned and concentrated supernatant was subjected to ultracentrifugation at 100,000× g for 2 h at 4 °C using the OptimaL-80 XP ultra-centrifuge equipment  to precipitate EVs. The precipitated EV products were washed using filtered phosphate-buffered saline (PBS), resuspended in PBS, and aliquoted to store at −80 °C until use. Total EV proteins were determined using a BCA protein assay kit following the manufacturer’s instruction.Previously established TRAIL-transduced human UC-MSCs (MSCflTs)  were useA nanoparticle flow cytometer   was usedg for 1.5 h to remove free dyes, and then resuspended in 100 kDa ultrafiltered PBS. A549 cells were first cultured in chamber slides for 24 h, and then co-incubated with DiI labeled EVs for 5 h and 11 h for endocytosis and intracellular tracking assay, respectively. Subsequently, treated A549 cells were washed by PBS, fixed with 4% paraformaldehyde and co-labelled with the cellular lysosome staining probe Lysotracker , and finally examined and imaged by confocal microscopy .EVs were labelled with the lipid membrane dye DiI  for cellular uptake and tracking assay. EVs (60 µg) were first stained with 12 µM DiI for 25 min at RT in the dark. Then, labelled EVs were precipitated by ultracentrifugation at 100,000× g for 1.5 h to remove free dyes, resuspended in 100 kDa ultrafiltered PBS, and designated EV-DiR. Free DiR and EV-DiR were aerosolized and administered to lung tumor-bearing mice by inhalation, with 4 mice per group. Six hours post inhalation, mice were culled and their organs were isolated, including heart, liver, spleen, lung, kidney, and intestine for examination of DiR biodistribution by an in vivo imaging system  and recorded with a built-in CCD camera, respectively. The DiR fluorescence signal intensity was determined on the regions of interest and quantified using the Living Image software .EVs were labelled with the lipid membrane dye DiR  to examine their in vivo biodistribution in lung tumor-bearing mice. EVs (200 µg) were first stained with 10 µM DiR for 30 min at RT in the dark. Then, labelled EVs were precipitated by ultracentrifugation at 100,000× V/V) as the mobile phase and at a flow rate of 0.8 mL/min. Dina elution was monitored using the UV-Visible DAD detector at 254 nm. A serial Dina concentration was measured to establish a detection standard curve, including 0.2-, 0.5-, 1.0-, 2.0-, 5.0-, and 10.0 µg/mL.The potent cyclin-dependent kinase inhibitor Dinaciclib  was purchased from Selleck Chemicals  and dissolved in DMSO to prepare 10 mM stock solution. High-performance liquid chromatography (HPLC) was used to detect and measure Dina in solution using a C18 column  at 40 °C in isocratic mode with acetonitrile in water , via amphotericin B (AmB), or by sonication. The co-incubation was conducted to mix Dina and EV-T solution and incubate for 1 h at 4 °C. The AmB method consisted of adding 5% Amphotericin B  into the mixture of EV-Ts and Dina for co-incubation for 1 h at 4 °C. The sonication loading was performed by treating the Dina and EV-T mixture with sonication using a sonic dismembrator (Model 505). The sonication was set as the following: 20% amplitude, 6 cycles of 30 s on/off, with a 2 min cooling interval between each cycle. After sonication treatment, the sample was incubated for 1 h at 37 °C to restore the EV membrane integrity. EV-Ts with Dina loading treatment were subjected to ultracentrifugation at 100,000× The encapsulated Dina was quantitated by measuring the UV absorption at 254 nm using an ultraviolet spectrophotometer. A standard curve of Dina/UV absorption was first established using the 254 nm absorbance as a function of Dina concentration (µg/mL). Afterwards, the EV-T-Dina solution (200 µL) was first evaporated at 50 °C using a vacuum concentrator, and then supplemented with acetonitrile (200 µL) and treated by sonication to disrupt EV membrane and completely release encapsulated Dina. Subsequently, the sonicated EV-T-Dina solution was precipitated by using the EV precipitating agent , and then Dina-containing supernatant was harvested for determination of Dina concentrations using the standard curve of Dina.The therapeutic solution was converted to aerosol for inhalation therapy using a portable pressurized nebulizer . This nebulizer is designed to produce aerosolized droplets with a mean median diameter (MMD) of 2.2 µm, which is suitable for the inhalation-based treatment of lung cancer. An aerosol particle size analyzer  was used to determine the nebulized particle size and size distribution. The injection cell was first inserted into the analyzer and warmed up for 4 h at 37 °C. Then, the air flow meter and the high-energy vacuum pump were connected, and the air flow rate was set as 30 L/min. Subsequently, the standard operating procedure (SOP) was set using the associated software  as follows: test mode—fast mode; particle shading coefficient—1.33; dispersion medium—air. Next, the size analyzer was connected to the nebulizer and performed the size analysis of nebulized particles and droplets.DR) was calculated using the previously reported formula [Drug dialysis was carried out to assess and compare the stability of EV-T-Dina and free Dina. Both free Dina and EV-T-Dina were first dissolved in 2.0 mL of filtered PBS (pH 7.4) as 25.0 µg/mL in dialysis bags. Then, dialysis bags were incubated in 25 mL of PBS that was vortexed at 37 °C in a 50 mL flask in the dark. Every 1.0 mL of PBS was removed from the flask at various time points to measure released Dina by HPLC. The cumulative Dina release .The cytotoxicity of therapeutic agents was assessed by both cellular viability evaluation and apoptosis assay on A549-Luc, NCI-H23, and MSC cells following the previously established procedures . In brieCellular or EV proteins of interest were detected by immunoblotting according to previous description . In brieAn orthotopic lung tumor model was established and used to evaluate the therapeutic efficacy of aerosolized EVs in BALB/c nude mice , which were obtained from an SPF Biotechnology Company . The experimental procedures and protocols were approved by the Animal Ethics Committee of South China University of Technology . Animals were housed in pathogen-free animal facility with filtered air, autoclaved water, and food available all the time.7 cells/mL) and used for the preparation of an A549-Luc-containing Matrigel matrix (4.35 mg/mL) . Mice were first anesthetized and placed in the right lateral decubitus position. Then, 1 mL syringes with 29 gauge needles were used to percutaneously implant A549-Luc cells in Matrigel  into the right lateral thorax at the lateral dorsal axillary line, which is about 1.5 cm above the lower rib line and just below the inferior border of the scapula [® Lumina II Imaging System . Then, 20 mice were randomly divided into four experimental groups with 5 animals per group: vehicle control (Ctrl), Dina treatment (Dina), EV-T treatment (EV-T), and EV-T-Dina treatment (EV-T-Dina). Aerosol delivery of therapeutics was performed for approximately 30 min/day for 7 times in total for each mouse—with an interval of 2 days between treatments—using a portable nebulizer  [Luciferase-expressing A549 cells (A549-Luc) were resuspended in PBS (5 × 10 scapula . On day Germany) . The dosTwenty-four hours after the last treatment, mice were sacrificed and organs including heart, liver, spleen, kidney, and lung were resected for immuno-histochemistry (IHC) analyses following the procedures described previously . TUNEL at-test, and comparison among multi-groups was performed using one-way ANOVA/Bonferroni multiple-comparison post hoc correction. Significant probability values were indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.The software GraphPad Prism 8.0 was used to analyze data . Significant differences between two groups were analyzed using Student’s Dina and TRAIL were previously shown to synergize to induce apoptosis in cancer cells . Therefog to precipitate the EVs  by TRAIL-overexpressing cells ,18. So,  the EVs a. The is the EVs b, with aytometry c. The TRytometry d. The TRytometry e.2 = 0.9992) (A high-performance liquid chromatography (HPLC) analysis was performed to isolate Dina and the built-in UV spectrometer was used to measure Dina concentration, showing a detection peak at 11.451 min f. The de 0.9992) g.To prepare the EV-based aerosol for lung cancer treatment, we first needed to encapsulate Dina into the EV-Ts to produce complexed EVs that carry both TRAIL and Dina, a composite which has been designated EV-T-Dina. Three loading methods were tested for Dina’s encapsulation into the EV-Ts, which were mixing, sonication, and amphotericin B mediated approach. As shown in Free Dina in solution can be directly measured by using a UV spectrometer. However, the encapsulated Dina needs to be released from the EVs by sonication in acetonitrile for an accurate quantification. The loading rates via sonication were tested for different combinations of 100 µg/mL of EV-Ts with various Dina concentrations (1.0–100 µM), and the encapsulated Dina contents appeared proportional to the initial Dina concentrations. However, the Dina loading rates remained around 20.6% for various Dina loading concentrations. Therefore, for various Dina loading concentrations , the encapsulated Dina contents were measured and approximately calculated as 0.824–82.4 ng/µg EV-Ts.Free Dina (100 µM) and EV-T-Dina (20 µM) were incubated at 37 °C for 0–150 min, respectively, to test the stability of Dina in vitro. As shown in A drug release test was performed to characterize the EV-encapsulated Dina in vitro. As shown in In order to determine the in vivo stability and pharmacokinetics of Dina, two groups of 6-week-old female BALB/c nude mice were injected intraperitoneally with free Dina at the dose of 100 mg/kg or EV-T-Dina at the dose of 100 mg/kg (corresponding to encapsulated Dina 8.24 mg/kg), respectively. After 0.5–8.0 h of injection, the blood samples were collected, and the concentration of serum Dina was determined by HPLC. As shown in An analysis of the cellular uptake of the EVs was performed in A549 cells to examine the plausibility of using EVs for drug delivery. As shown in Next, the cytotoxicity of EV-T-Dina was examined in two highly TRAIL-resistant NSCLC lines (NCI-H23 and A549) and the normal MSC cells. Low doses of TRAIL from EV-T (0–4.0 ng/mL) and low doses of Dina encapsulated in EV-T (0–40 nM) were combined to perform a cell treatment for 24 h, followed by cell proliferation and viability assays. As shown in According to the cytotoxicity results, 2 ng/mL EV-T TRAIL and 20 nM Dina were determined as optimal drug combination doses for an apoptosis assay in both A549 and MSC cells to further evaluate the anticancer activity of EV-T-Dina. As shown in cFLIP, MCL-1, and Survivin are three well-known intrinsic inhibitory factors in the TRAIL signaling pathway . The proCollectively, these data demonstrate that EV-T-Dina is highly efficient for inducing apoptosis in the TRAIL-resistant A549 line with a mechanism associated with the concomitant downregulation of cFLIP, MCL-1, and Survivin.Therapeutic solutions were converted into aerosols by using a portable pressurized nebulizer. The appropriate insert was chosen for the nebulizer, aiming to mainly produce an aerosol particle size below 5.0 µm in diameter. This size was chosen to ensure that the therapeutic aerosols could be inhaled into the bronchioles and alveoli.A laser diffraction system was used to measure the size distribution of aerosol particles and droplets. As shown in Free DiR and DiR-labelled EV-T-Dina (EV-T-Dina/DiR) were aerosolized and inhaled by the mice to examine their in vivo biodistributions. The mice were sacrificed 6 h post inhalation, and their organs were excised and examined to determine their DiR distribution. As shown in Having seen the synergistic apoptosis induction in the A549 cells by EV-T-Dina and established the unique distribution of the inhaled EV-T-Dina aerosol in the lungs, the therapeutic potency of the aerosol was next evaluated in an orthotopic lung tumor model in vivo, as shown in Luciferase-expressing A549 cells (A549-Luc) were implanted into the lungs of nude mice to induce lung tumor development. Three weeks post implantation, the mice were examined for luciferase bioluminescence, followed by randomization to assign animal treatment groups with comparable bioluminescence and thus similar initial tumor burdens in test groups. Four inhalation treatment groups were tested, including vehicle control, EV-T, Dina, and EV-T-Dina and seven treatments were performed in total for each mouse using a pressurized nebulizer in a 2-week treatment cycle. The treatment results were determined by bioluminescence imaging in vivo and are shown in The animals’ lung tumors were subsequently removed and assessed for the cell proliferation marker ki67 and cleaved (activated) caspase-3 expression by IHC staining at the experimental end point. As shown in Moreover, mouse organs including heart, liver, spleen, and kidney were collected from both the vehicle and EV-T-Dina inhalation groups at the experimental endpoint and evaluated for apoptosis by TUNEL staining . This waCollectively, these observations demonstrate that the inhalation of EV-T-Dina aerosol constitutes a novel therapy, which is highly effective and safe for lung cancer treatment.In this study we have shown: (1) that Dina can be encapsulated into TRAIL-expressing EVs to produce the complexed agent EV-T-Dina, (2) that integrated Dina and TRAIL are synergistic for the specific killing of cancer cells with high efficiency, (3) that EV encapsulation improves Dina stability both in vitro and in vivo, (4) that EV-mediated aerosol delivery showed both a unique distribution and an improved drug retention in the lungs, (5) and that the aerosol delivery of EV-T-Dina demonstrated high efficacy and good safety for lung cancer treatment.Accounting for about 85% of all types of lung cancer, NSCLC are generally treated by surgical resection in combination with systemic chemotherapy either preoperatively or postoperatively. Although intending to find a cure, the therapeutic efficacy of such a treatment strategy is frequently affected by chemotherapy resistance in NSCLC. EGFR-targeting agents are primarily used as first-line drugs for the treatment of EGFR-mutant NSCLC, such as erlotinib, gefitinib, and afatinib. These drugs are beneficial for extending the life spans of patients but possess limited effectiveness due to drug resistance. Moreover, these first-line treatments are often associated with adverse side effects, such as an elevation in serum aminotransferase levels during therapy, indicating apparent liver toxicity.As a second generation of CDK inhibitor and an experimental drug, Dina SCH-727965) has been widely tested in phase II clinical trials for treating advanced breast cancer 965 has b, multiplThe pulmonary delivery of therapeutics has been employed to treat all types of lung diseases  includinEVs are an emerging novel drug delivery system with many advantages , such asThe observed low stability of free Dina solution at 37 °C may provide evidence supporting the previously examined short plasma half-life of Dina in mice , given tChemotherapy is a common cancer treatment approach with some inevitable shortcomings, such as low bioavailability, poor tumor tropism, a short plasma half-life, and systemic adverse side events, frequently resulting in the onset of drug resistance in tumors . As showPreviously, we found that TRAIL-expressing EVs (EV-T) can be combined with Dina to produce synergistic anti-tumor activity . Dina alIn this study, we further demonstrated that Dina and TRAIL can be integrated into EVs to produce the complexed agent EV-T-Dina, which is highly effective towards apoptosis induction in cancer lines. More importantly, EV-T-Dina can be delivered into lungs by aerosol inhalation, demonstrating a unique lung distribution and good safety. This strategy has several advantages, such as the lung enrichment of EV aerosols, the synergistic action of the combined drugs, the improved solubility and stability of Dina, and an improved therapeutic efficacy and safety. This can be an example to be used towards the pulmonary route delivery of other therapeutic agents alone or in combination. To the best of our knowledge, this is the first time that the EV-based aerosol has been prepared and tested for the treatment of lung cancer.In this study, Dina, after its encapsulation in EV-T, was administered through inhalation at a low dosage and was shown to drastically induce augmented apoptosis in A549 lung cancers without any observed adverse side events, suggesting the plausibility of using the EV-based aerosol delivery of combined therapeutics for better efficacy and safety . In the This study has demonstrated the plausibility of developing a human lung cancer inhalation therapy based on drug delivery by EVs. However, some concerns remain to be clarified. First, the therapeutic efficacy revealed in an experimental lung cancer model may not guarantee a comparable effectiveness in human diseases. Second, the therapeutic safety for human lung cancer needs to be clarified, although a promising treatment tolerability has been observed in this study. Third, the combination of TRAIL and Dina was only tested for being highly effective for the specific killing of two lung cancer lines, H23 and A549; next, the effectiveness of this combined therapy needs to be extended to a broader range of lung cancer lines. Fourth, a patient selection criterion may need to be established for the precision application of the therapy. Therefore, what we have surely demonstrated now is the plausibility of using EVs for the lung tissue-targeted delivery of therapeutics. Importantly, this study rationalizes further investigations of the EV-mediated pulmonary delivery of various therapeutic agents, alone or in combination, for the treatment of lung diseases.In conclusion, chemotherapies such as Dina can be encapsulated in genetically engineered anti-cancer EVs, such as EV-T, to produce complexed inhalation agents such as EV-T-Dina, which concomitantly suppress the expression of innate apoptosis inhibitors such as cFLIP, MCL-1, and Survivin in cancer cells, thereby improving the therapeutic potential and treatment safety of lung cancer therapeutics.


BRASSINOSTEROID INSENSITIVE 2 (BIN2) alleles as decision mutants with “confused” phenotypes. An assessment of organ and cell length suggested that hypocotyl elongation occurred predominantly via cellular elongation. In contrast, root growth appeared to be regulated by a combination of cell division and cell elongation or exit from the meristem. Gain- or loss- of function bin2 mutants were most severely impaired in their ability to adjust cell geometry in the hypocotyl or cell elongation as a function of distance from the quiescent centre in the root tips. This study describes a novel paradigm for root growth under limiting conditions, which depends not only on hypocotyl-versus-root trade-offs in the allocation of limited resources, but also on an ability to deploy different strategies for root growth in response to multiple stress conditions.Plants often adapt to adverse conditions via differential growth, whereby limited resources are discriminately allocated to optimize the growth of one organ at the expense of another. Little is known about the decision-making processes that underly differential growth. In this study, we developed a screen to identify decision making mutants by deploying two tools that have been used in decision theory: a well-defined yet limited budget, as well as conflict-of-interest scenarios. A forward genetic screen that combined light and water withdrawal was carried out. This identified  The ability to grow in response to limiting, adverse conditions is a survival strategy unique to plants. This study addresses the tight regulation of differential growth in plants, and how growth is achieved under multiple stress conditions, in the absence of a carbon or energy source. We design a screen to identify decision-making mutants by deploying two tools that have been used in decision theory: a well-defined yet limited budget, as well as conflict-of-interest scenarios. We also apply a new combination of stress factors: water stress and light deprivation. Our manuscript addresses tradeoffs in hypocotyl versus root growth, with an emphasis not on growth arrest but rather on enhanced growth responses to abiotic stress factors. Our findings challenge the widely accepted view that root growth correlates with meristem size; we propose alternative root growth strategies for adaptation to adverse, limiting conditions. Our genetic screens have identified the BIN2 clade of shaggy-like kinases as playing a central role in decision-making in the seedling. We address the controversy in the literature regarding brassinosteroid function at a cellular level. This we do by exploring the role of BR signalling in the regulation of cell elongation and geometry in response to abiotic stress cues. Our findings suggest that BIN2 and its homologues are required for the decision as to which growth strategy to adopt under different environmental conditions. Plants often adapt to adverse conditions via differential growth, whereby limited resources are differentially allocated to optimize the growth of one organ at the expense of another. A good example of this is the etiolation response , in whicPotentilla reptans, which was shown to adopt one of three strategies–vertical growth, shade tolerance or lateral avoidance–to optimize above ground responses to prevailing light-competition scenarios ,  action; ,74). ThuFuture experiments will address where in the seedling decision-making processes occur, the nature, interdependence and movement of the acro- and basipetal signals, and how these facilitate shoot to root communication to fine tune trade-offs between root versus shoot growth. 3D imaging will be required to assess the impact of abiotic stress and/or of BR signalling on different cell files or tissue layers in the root  K, N or P and incubated for seven days in the dark. (A-D) germination in the absence of a carbon source. Panels (A-D) are from the same experiment. Minor fluctuations were detected, but no clear trade-offs between hypocotyl and root growth were observed when nutrient stress was applied to dark-grown seedlings. Based on the long hypocotyls (A) and short roots (B), there is a clear priority for light (translating into hypocotyl growth) over nutrients (translating into root growth) in the germinating seedling as seen in the hypocotyl/root ratio (C) and the total length (D). (E-H) germination in the presence (+) of 1% sucrose as a carbon source. The presence of sucrose as a carbon source increased the total length of the seedlings up to two-fold (h); numbers above the columns are fold-changes compared to the same condition without sucrose.  Panels in  depict the impact of a carbon source. In the presence (+) of a carbon source, hypocotyl length remained fairly constant but the roots were able to grow longer  of sucrose). Thus, there was no clear trade-off, by which we refer to the growth of one organ at the expense of another. As the genetic screen was designed to mimic limiting conditions, we omitted sucrose from the media in all further experiments. The number (n) of seedlings measured per condition is in grey below the graph. (TIF)Click here for additional data file.S2 FigFig 1.Hypocotyl (A) and root (B) lengths of seedlings (Ler wild type) germinated under osmotic (100mM mannitol) or salt stress (50-100mM NaCl) in the dark in the absence of a carbon source. No reproducible differences in the ratio (C) were observed (P > 0.5). The seedling total length (D) decreased with increasing salt concentration. The number (n) of seedlings measured per condition is in grey below the graph. P-values were computed with a two-tailed student’s T-test and are represented as follows: *: 0.05–0.01; **: 0.01–0.001; ***: 0.001–0.0001; ****: 0.0001–0.00001, *****: < 0.00001. Related to (TIF)Click here for additional data file.S3 FigFig 1D): water stress applied in the dark increases root length at the expense of hypocotyl length, giving rise to a decrease in the hypocotyl/root ratio. (C) The length of wild type (Col-0) seedlings was seen to decrease with increasing water stress after seven , and this decrease was observed in control experiments to be due to delayed germination induced by water stress. (D) Roots were stained with methanol blue; arrows point to the junction between the hypocotyl and root and arrowheads to the end of the root. The number (n) of seedlings measured per condition is in grey below the graph. P-values were computed with a two-tailed student’s T-test and are represented as follows: *: 0.05–0.01; **: 0.01–0.001; ***: 0.001–0.0001; ****: 0.0001–0.00001, *****: < 0.00001. Related to Fig 1 and S5 Fig.(A) Hypocotyl/root ratio of seedlings (Col wild type) germinated in the light with (lightW) or without (light) water stress (PEG -0.4MPa); the clearest response to water stress in the light is a reduction in hypocotyl length (left panel). This is consistent with the observations of van der Weele et al.  on the u(TIF)Click here for additional data file.S4 Fig-2 s-1. (B) Red light at varying intensities ranging from 4.2 to 0 μmol m-2 s-1. (C) Far-red light at 0.5 μmol m-2 s-1, compared to dark grown seedlings. A decreasing intensity gradient of red light, as well as low levels of far-red light, increases hypocotyl length at the expense of root length as seen in the hypocotyl/root ratio. (D-G) Quadruple cry1 cry2 phyA phyB mutant seedlings  had no hypocotyl response to light  but had highly significant responses to water stress applied in the dark (G); corresponding Ler wild type . The number (n) of seedlings measured per condition is in grey below the graph. Benjamini–Hochberg corrected P-values are represented as follows: *: 0.05–0.01; **: 0.01–0.001; ***: 0.001–0.0001; ****: 0.0001–0.00001, *****: < 0.00001. Related to (A-C) Wild-type (Col-0) seedlings were germinated on MS media under different light conditions, with or without water stress. (A) Blue light at varying intensities ranging from 5.3 to 0 μmol m(TIF)Click here for additional data file.S5 FigP-values were computed with a two-tailed student’s T-test and are represented as follows: **: 0.01–0.001; ***: 0.001–0.0001; ****: 0.0001–0.00001, *****: < 0.00001. Scale bars = 1mm. See related Figs 1 and S3.Wild-type seeds (Col-0) were germinated on ½ MS in the dark with or without water stress. (A) Hypocotyl and root lengths, as well as hypocotyl/root ratio. Note that seedlings germinated under water stress in the dark have shorter hypocotyls and longer roots than in the dark, giving rise to a decreased hypocotyl/root ratio. (B) Hypocotyl width; hypocotyls are wider in light than in the dark; water stress in the dark results in a decrease in hypocotyl width. (C) Hypocotyl volume; the volume increases in the dark (cf. light) and decreases in response to water stress in the dark. The number (n) of seedlings measured per condition is in grey below mean ±StDev bar graphs. (TIF)Click here for additional data file.S6 Figratios (see main bin2-1. (C) The total length is depicted as the hypocotyl  and root . B1 was shorter than Ler (L) in the light and in the dark, but not under darkW conditions. The number (n) of seedlings measured per condition is in grey below the graph. P-values were computed with a two-tailed student’s T-test and are represented as follows: *: 0.05–0.01; **: 0.01–0.001; ***: 0.001–0.0001; ****: 0.0001–0.00001, *****: < 0.00001. Related to Fig 2.(A) Light responses were attenuated in B1, with a shorter hypocotyl and longer root in the dark. (B) RQsee main  were com(TIF)Click here for additional data file.S7 FigS1 Table; null alleles are depicted in regular font, semi-dominant or dominant in bold and higher order mutants are underlined. Datasets are as in Fig 3, with the exception of bzr1-1D and bri1-116 bzr1-1D, where different representative replicates are shown.Violin plots of the hypocotyl, root and ratio responses of BR pathway mutants, with the corresponding wild-type ecotype as reference. The dot represents the mean and the line the 95% confidence interval. Note the high variance of the wild-type (Col-0) root response under darkW. Mutant alleles are described in (TIF)Click here for additional data file.S8 FigX-axis and the P-Value of the response on the Y-axis . Null alleles are depicted in regular font, semi-dominant or dominant in bold and higher order mutants are underlined. (A) Hypocotyl responses to dark versus darkW conditions. Note that bri1-116 bzr1-1D and the semi-dominant bin2-1 have an attenuated hypocotyl response, RQhypocotyl (magenta arrows). This was not observed in the triple bin2-3bil1bil2 knock out (green arrow). (B) Root responses to dark versus darkW conditions. Note that the triple bin2bil1bil2 knock out has the strongest RQroot phenotype (magenta arrow). This is in contrast to bin2-1 (green arrow). Thresholds used to interpret the results are tabulated at the bottom of the figure; magenta colour indicates an attenuated and green an exaggerated response. Related to Figs 4.Response quotients  and volcano plots (right) of the hypocotyl and root responses water stress in the dark. RQs are normalized to the wild-type ratio quotient; a value of 1  indicates that the response to a shift from dark to darkW is similar to that of the respective wild-type ecotype. Each replicate is represented by a dot; purple dots are for initial and grey dots for optimized screen conditions; blue dots are for data from SEM measurements. Volcano plots with the mean RQ depicted on the left on the (TIF)Click here for additional data file.S9 FigP-values were computed with a two-tailed student’s T-test and were non-significant: >0.05. Related to Figs 6.The number (n) of seed bags analysed per condition is in grey below mean ±StDev bar graphs. Each seed-bag contained on average 397 seed. (TIF)Click here for additional data file.S10 Figcpd. (C) BR perception mutant bri1brl1brl3 (D) BR signalling mutant bin2-1 . (E) bin2-3bil1bil2 triple knockout; (F) Transcription factor mutant bzr1-1D, a dominant allele. (G) Bypass mutant bri-116 bzr1-1D. (A-M) Null alleles are depicted in regular font, semi-dominant or dominant in bold and higher order mutants are underlined. Note that all mutants had significant responses to light versus dark conditions. At least 3 experiments were performed for each line, and a representative one is shown here on the basis of RQ and P values . The number (n) of seedlings measured per condition is in grey below the mean ±StDev bar graphs. P-values were computed with a two-tailed student’s T-test and are represented as follows: *: 0.05–0.01; **: 0.01–0.001; ***: 0.001–0.0001; ****: 0.0001–0.00001, *****: < 0.00001. For mean RQ values and median P-values see S11 Fig. Ecotypes are described in S1 Table. Related to Figs 4.Seedlings were germinated on ½ MS in the light and dark (A) Col-0 (wild type). (B) BR biosynthesis mutant (TIF)Click here for additional data file.S11 Figcpd, bri1brl1brl3 and bin2-1 mutants have a severely attenuated hypocotyl response RQhypocotyl. (B) Root responses to light versus dark conditions. Note that cpd, and bin2-1 mutants have the strongest RQroot phenotype, and that bin2-1 gain of function and bin2-3bil1bil2 loss of function mutants have opposite root phenotypes. (C) Hypocotyl/root ratio responses to light versus dark conditions. In all three volcano plots, cpd and bin2-1 mutants are the most severely impaired (most attenuated response (RQ), lowest P-value). Thresholds used to interpret the results are tabulated at the bottom of the figure; magenta colour indicates an attenuated and green an exaggerated response. Related to Figs 4.Response quotients  and volcano plots (right) of the hypocotyl, root or ratio responses to light versus dark. RQs are normalized to the wild-type ratio quotient; a value of 1  indicates that the response to a shift from light to dark is similar to that of the respective wild-type ecotype. Each replicate is represented by a dot; grey dots are for optimized screen conditions; blue dots are for data from SEM measurements. Volcano plots with the mean RQ depicted on the left on the X-axis and the P-value of the response on the Y-axis . Null alleles are depicted in regular font, semi-dominant or dominant in bold and higher order mutants are underlined. (A) Hypocotyl responses to light versus dark conditions. Note that (TIF)Click here for additional data file.S12 FigUBIQUITIN-PROTEIN LIGASE-LIKE PROTEIN as a reference for normalization (see S7 Method). Expression in the light was set at 100% for both genotypes. Gene expression was significantly downregulated in the dark, in both the wild type and in bin2-3bil1bil2 mutants. Data represent means ± StDEV of at least three independent experiments, where each measurement was based on three technical replicates. Three independent lines were used for the mutant. P-values were computed with a two-tailed student’s T-test and are represented as follows: **: 0.01–0.001; *****: < 0.00001.Seed were germinated on 1/2 MS plates and incubated in the light  or dark  for 10 days. Transcript abundance was determined by qRT-PCR with (TIF)Click here for additional data file.S13 Figbin2-3bil1bil2 triple knockout; (B) Transcription factor mutant bzr1-1D, a dominant allele. The number (n) of seedlings measured per condition is in grey below the graph. P-values were computed with a two-tailed student’s T-test and are represented as follows: *****: < 0.00001 (C) RQhypocotyl response quotient of the hypocotyl under light/lightW conditions, normalized to the wild-type response quotient; a value of 1  indicates that the response to a shift from light to lightW is similar to that of the respective wild-type ecotype. Each replicate is represented by a dot. (D) Volcano plot with the mean RQhypocotyl depicted in (C) on the X-axis and the median P-Value of the response on the Y-axis . Related to Figs 4.Hypocotyl responses of (A) (TIF)Click here for additional data file.S14 Figbin2-3bil1bil2 triple null BR signalling mutant. See S8 Method.As described by Fridman et al.  we used (TIF)Click here for additional data file.S15 FigP = 1.2E-08) and a concomitant decrease in meristem size. (B) In the dark, water stress results in a decrease in the number of transition cells . The number (n) of seedlings measured per condition is in grey below the mean ±StDev bar graphs. P-values were computed with a non-parametric Mann-Whitney-U tests with a Benjamini–Hochberg correction in (C) and with a two-tailed student’s T-test in panels A, B, E, F; they are represented as follows: *: 0.05–0.01; **: 0.01–0.001; ****: 0.0001–0.00001; *****: < 0.00001. Scale bars: 50 μm. Related to Fig 6.(A) A shift from light to darkness decreases the number of isodiametric cells Click here for additional data file.S16 Figbin2-3bil1bil2 seed were germinated in the light (orange), dark (black) or dark with -0.4MPa water stress (blue). 10 days after incubation, single epidermal cell files were measured, starting at the epidermal/ lateral root cap initials. (A) Meristem size determined via mixed Gaussian models, as described . (B) Mature cell length, based on the ten most elongated cells for each condition.  Cell lengths (C) and width (D) of consecutive cells as a function of cell number from the quiescent centre (QC); the fitted lines were generated with Local Polynomial Regression Fitting with the ‘loess’ method in R and grey shading designates the 95 percent confidence interval. The purple arrows point to the relatively flat slope for cell length  in the darkW condition (C). The sample size (n) is given as the number of seedlings in panels a and b and as the number of cells/ number of seedlings that were analysed in panels C, D. P-values were computed with a two-tailed student’s T-test and are represented as follows: *: 0.05–0.01; ***: 0.001–0.0001; *****: < 0.00001. Related to Fig 6.scribed Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Method(PDF)Click here for additional data file.S2 Method(PDF)Click here for additional data file.S3 Method(PDF)Click here for additional data file.S4 Method(PDF)Click here for additional data file.S5 Method(PDF)Click here for additional data file.S6 Method(PDF)Click here for additional data file.S7 Method(PDF)Click here for additional data file.S8 Method(PDF)Click here for additional data file. 21 Sep 2022Attachmentrenamed_64395.pdfSubmitted filename: Click here for additional data file. 12 Oct 2022Dear Dr Assaad,Thank you very much for submitting your Research Article entitled 'A role for brassinosteroid signaling in decision-making processes in the Arabidopsis seedling.' to PLOS Genetics.The manuscript was fully evaluated at the editorial level and by independent peer reviewers. 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You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.Please let us know if you have any questions while making these revisions.Yours sincerely,Claudia KöhlerSection EditorPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The study is timely and important to understand how plants cope with manifold stimuli to adjust growth by orchestrating cell elongation rate. All my concerns were addressed, and I want to endorse the study for publication.Reviewer #2: This manuscript by Kalbfuß et al identifies a role for brassinosteroid signaling in a decision-making process of root vs shoot growth under the combination of darkness and water stress. We reviewed a previous version of this manuscript and the authors have now adequately addressed many of our concerns. They provided new data in the form of BR mutant analysis and interesting observations showing that the increase in root length in response to water deficit in the dark appears to operate through increased cell divisions despite the smaller meristem size. Overall the revised manuscript is solid and well written. We have several relatively minor comments that should be addressed which are listed below.1. Given the lot-to-lot and plate-to-plate variability that the authors note with PEG assays, can they be confident in claiming that variable hypocotyl versus root lengths are a read-out for a “confused phenotype”? It’s not clear that this claim is essential in their story demonstrating the root-versus-shoot tradeoffs mediated by BRs.2. Both gain-of-function (bin2-1) and loss-of-function alleles (bin2-3bil1bil2) of BIN2 seem to be affected by water deficit in the dark in a similar direction , which could be further discussed.3. Pg 12 line 19 refers to bri1-116 bzr1-1D double mutants: “In this line, the BR pathway, BIN2 function within this pathway, is effectively bypassed”. BIN2 activity is not directly affected by the addition of bzr1-D in the double mutant, but BZR1, downstream of BIN2, is constitutively active regardless of the BR signal. Other BIN2 substrates may still be phosphorylated and regulated by BIN2 which would be active in the bri1 background.4. The authors claim: “In addition, an analysis of root meristem properties in bin2-3bil1bil2 shows that BR signaling is required for the Arabidopsis seedling’s ability to deploy different root growth strategies in response to abiotic stress cues under limiting conditions.” While I agree that bin2-3bil1bil2 shows interesting changes in root morphology and response to combined darkness/water stress, the fact that this mutant has increased BR signaling as the result of loss of BIN2 and homologs (negative regulators) makes it difficult to claim that this genetic analysis indicates a requirement for BR signaling in this context. The loss of function mutants in the pathway  would be more appropriate to support the essentiality of BR signaling.Reviewer #3: The revised manuscript clearly improved. Most comments in the first submission have been addressed and new data was added. Nevertheless, a few issues are listed below:The focus on brassinosteroids improves the manuscript. However, the mechanistic insights how brassinosteroid are involved in decision making, remain largely obscure. As presented by the data in fig 3 and 4, I would even suggest that the canonical BR-signaling via the BZR1/BES1 signaling is not involved in the “decision-making” as the gain of function mutants bzr1-1D and bes1-D responded like wild type and the bri1-116 bzr1-1D double mutant showed no rescue; BIN2 is known to branch out to integrate other signaling pathways, thus BIN2 possibly operates via one of these pathways. I understand that identifying the mechanism downstream of BIN2 is out of the scope of this work; because of this the claims made on the role of BIN2 should be done very carefully.There is also an inconsistency on the use of mutants in newly added experiments that should be addressed: hypocotyl cell parameters are from bin2-1, a gain-of-function mutant, while the new data on root meristem and root cell geometry come from bin2-3bil1bil2, a triple loss-of-function mutant. To draw conclusions the gain-of-function and loss of function mutants should be both included.In Fig. 5d the representative images of bin2-1, at least in my eyes, show a stronger reduction of hypocotyl cell length in darkW compared to dark than shown in the graph of 5e.The new data on root meristem properties is very interesting.The summary in Fig. 7 could also be moved into the supplements since the data is presented in Fig. 5 and 6.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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Neonatal painful procedures causes acute pain and trigger long-term changes in nociceptive processing and anxiety behavior, highlighting the need for adequate analgesia during this critical time. Spinal serotonergic receptors 5-HT1a and 5-HT3 play an important role in modulating incoming nociceptive signals in neonates. The current study aims to attenuate acute and long-term hypersensitivity associated with neonatal procedural pain using ondansetron (a 5-HT3 antagonist) and buspirone (a 5-HT1a agonist) in a well-established rat model of repetitive needle pricking. Sprague-Dawley rat pups of both sexes received ondansetron (3 mg/kg), buspirone (3 mg/kg) or saline prior to repetitive needle pricks into the left hind-paw from postnatal day 0–7. Control animals received tactile stimulation or were left undisturbed. Acute, long-term, and post-operative mechanical sensitivity as well as adult anxiety were assessed. Neonatal 5-HT1a receptor agonism completely reverses acute hypersensitivity from P0-7. The increased duration of postoperative hypersensitivity after re-injury in adulthood is abolished by 5-HT3 receptor antagonism during neonatal repetitive needle pricking, without affecting baseline sensitivity. Moreover, 5-HT1a and 5-HT3 receptor modulation decreases adult state anxiety. Altogether, our data suggests that targeted pharmacological treatment based on the modulation of spinal serotonergic network via the 5-HT1a and 5-HT3 receptors in neonates may be of use in treatment of neonatal procedural pain and its long-term consequences. This may result in a new mechanism-based therapeutic venue in treatment of procedural pain in human neonates. Newborns admitted to the neonatal intensive care unit (NICU) can be exposed to repetitive nociceptive input during a vulnerable time frame for the central nervous system, as part of their care and treatment. The neonatal period is unique in how it processes nociceptive stimuli, and the nociceptive network undergoes developmental changes in early life –3. Due tSerotonin  is a key modulator of anxiety behavior and perception of pain, and its ascending and descending fibers are distributed throughout the brain and spinal cord . VirtualDescending serotonergic modulation of the spinal nociceptive network, originating in the rostral ventromedial medulla (RVM), is immature in early life and undergoes developmental changes in anatomy and functionality. Importantly, the functionality switches from facilitation to inhibition of nociceptive signaling in early life , 27. SinDuring the neonatal period, facilitation of nociceptive signaling is mediated via the 5-HT3 receptors (R) in the dorsal horn –30 whereThe aim of this study is to prevent the acute and long-term consequences of repetitive neonatal procedural pain by pharmacological targeting the serotonin 5-HT1aR or 5-HT3R in neonates. Here, we used buspirone, a full agonist of the 5-HT1aR to produce inhibition , or ondaAll experiments and procedures were performed in line with the European Directive for Protection of Vertebrate Animal Used for Experimental and Other Scientific Purposes (2010/63/EU), and were ethically approved by the Committee for Experiments on Animals, Maastricht University, Maastricht, the Netherlands (DEC protocol 2017-017). Adult Sprague-Dawley rats were purchased from Charles River Laboratory , and were mated at Maastricht University. Animals were housed under reversed day-night conditions , in temperature (21 ± 1°C) and humidity (55 ± 15%) controlled rooms with food and water available ad libitum. Since the circadian rhythm of animals seems capable of affecting pain sensitivity, especially mechanical sensitivity [reviewed in ], all ben = 12), 2. NP with buspirone treatment , 3. NP with ondansetron treatment  or 4. repetitive tactile stimulation . A separate litter was left completely undisturbed . Rat pups (conditions 1–3) were noxiously stimulated with repetitive needle pricks (NP) four times a day at 09:00 a.m., 10:00 a.m., 11:00 a.m. and 12:00 p.m. from P0 to P7 as previously described (On the day of birth (P0) newborn pups were randomized, using a computer-generated randomization list, to one of four conditions; 1. repetitive needle pricking  was dissolved in sterile saline in a concentration of 2 μg/μl. For 5-HT1aR agonism, the selective agonist Buspirone  was dissolved in sterile saline to a concentration of 10 μg/μl. Pilot studies testing 1, 3 and 5 mg/kg BUS and 0.3, 1 and 3 mg/kg OND showed that 3 mg/kg BUS and 3 mg/kg OND were most promising in preventing acute hypersensitivity associated with neonatal repetitive needle pricking see , withoutTo assess the effect of neonatal 5-HT1aR agonism and 5-HT3R antagonism on general postnatal development, physical developmental milestones such as pinna detachment , incisor eruption (of lower and upper teeth), fur development, complete opening of both ears and complete opening of both eyes were expressed as the number of postnatal days required for the appearance of these milestones . These mTo assess acute post-operative hypersensitivity, a plantar hind-paw incision surgery (or Brennan model for postoperative pain) was performed on all animals . BrieflyMechanical sensitivity was assessed by determining the paw withdrawal thresholds (PWT) of the hind paws to calibrated Von Frey filaments. Briefly, animals were placed in a transparent box resting on an elevated mesh floor. After a 15-min acclimation period, a series of von Frey filaments  were applied to the plantar surface of the hind paw for 5 s using the up-down method . MechaniThe open field test was used to evaluate locomotor activity and anxiety-related behavior in adulthood (at 8 weeks of age) . BrieflyThe Elevated Zero Maze (EZM) was used to assess anxiety-related behavior . BrieflyP-value <0.05 was considered statistically significant. Mechanical force resulting in a 50% withdrawal frequency was assigned as the PWT and was calculated using a sigmoid curve fitting in Graphpad Prism 9. For the neonatal period, an area under the curve (AUC) analysis was performed. The AUC was calculated based on the PWTs for each group over the entire neonatal period (P 0–7) and statistically compared using a one-way ANOVA with Bonferroni post-hoc correction. Differences in PWT in the first neonatal week, mechanical sensitivity throughout development, and post-operative mechanical sensitivity were analyzed using a repeated measures analysis of variance (ANOVA) with Bonferroni post-hoc correction. Both within- and between-group analysis were run. A non-parametric Kruskal-Wallis tests was used to compare differences in developmental milestones between conditions. A two-way ANOVA was performed to compare the effects of condition and sex on anxiety-like behavioral measurements. For all analysis, the assumptions for statistical analysis were checked and met. For all behavioral analysis but the anxiety-like behavioral testing, the sex effect was tested and not significant, therefore data of both sexes was pooled to increase statistical power.All data are presented as mean ± SEM and plotted using Graphpad Prism 9 . A p > 0.05; p = 0.9951; p = 0.5928; Pinnae detachment, lower and upper incisor eruption, fur development, ear opening and eye opening are unaffected by neonatal condition  = 0.9085; p = 0.444; p = 0.4096; During the first postnatal week ipsilateral mechanical sensitivity increases over time, but not equally for all conditions . The PWT increase over time seems dependent upon neonatal condition for the ipsilateral , with UD animals showing lower contralateral PWTs at PI+7 as compared to NP, NP+BUS and NP+OND animals. The injury-induced change from baseline PWTs is not significantly affected by condition and only shows a significant effect of time .A significant interaction between time and condition is noted in contralateral PWTs  but is unaffected by neonatal condition .In the OFT, no significant effect of condition . Females do not show differences between conditions in the time spend in the open arms of the EZM  = 0.2669; p = 0.8963). In males, UD spend significant less time in the open arms of the EZM as compared to NP+BUS and NP+OND animals . The number of open arm entries in the EZM reveals a significant effect of sex  and condition [F  = 2.788; p = 0.0365; P = 0.0266).In the EZM, the percentage of time spend in the open arms of the EZM significantly differs between males and females [effect of sex: F  = 19.75; Neonatal repetitive needle pricking leads to acute hypersensitivity and increased duration of post-operative recovery after re-injury in adulthood –17, 23. The 5-HT3R and 5-HT1aR are important in a variety of neurodevelopment processes , 46. Thiin vivo to attenuate sensitivity after noxious stimuli in neonatal animals, suggesting the inhibitory effect of the 5HT1aR is present and functional during the neonatal period. This is important as descending serotonergic RVM-spinal dorsal horn have been reported to act in a facilitatory way in neonates .AK, EJ, JP, DT, and NH conceived the research. AK, EJ, and NH designed and conceptualized the research and contributed to its structure and content. AK performed the research, analyzed the data, and drafted the manuscript and figures. EJ provided the resources for this research. All authors commented on previous versions of the manuscript, critically revised, and quality assessed the manuscript. All authors have read and approved the final version of the manuscript.AK received financial support for this research provided by the Pain Knowledge Centre from Maastricht and an institutional grant from University Maastricht, School of Mental Health and Neuroscience.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Multisystem inflammatory syndrome is a severe manifestation of SARS-CoV-2 in children. The incidence of MIS-C after infection is poorly understood. There are very few cohorts describing MIS-C in Africa despite MIS-C being more common in Black children worldwide.A cohort of children with MIS-C and healthy children was recruited from May 2020 until May 2021 from the two main paediatric hospitals in Cape Town, South Africa. Clinical and demographic data were collected, and serum was tested for SARS-CoV-2 antibodies. The incidence of MIS-C was calculated using an estimation of population exposure from seroprevalence in the healthy group. Summary data, non-parametric comparisons and logistic regression analyses were performed.p = 0.002). The most common clinical features in MIS-C were fever (100%), tachycardia (98.5%), rash (85.3%), conjunctivitis (77.9%), abdominal pain (60.3%) and hypotension (60.3%). The median haemoglobin, sodium, neutrophil count, white cell count, CRP, ferritin, cardiac  and coagulation markers (D-dimer and fibrinogen) were markedly deranged in MIS-C. Cardiac, pulmonary, central nervous and renal organ systems were involved in 71%, 29.4%, 27.9% and 27.9% respectively. Ninety four percent received intravenous immune globulin, 64.7% received methylprednisolone and 61.7% received both. Forty percent required ICU admission, 38.2% required inotropic support, 38.2% required oxygen therapy, 11.8% required invasive ventilation and 6% required peritoneal dialysis. Older age was an independent predictor for the requirement for ionotropic support . The median hospital stay duration was 7 days with no deaths.Sixty eight children with MIS-C were recruited with a median age of 7 years . Ninety seven healthy children were recruited with a 30% seroprevalence. The estimated incidence of MIS-C was 22/100 000 exposures in the city in this time. Black children were over-represented in the MIS-C group (62% vs 37%, The lack of reports from Southern Africa does not reflect a lack of cases of MIS-C. MIS-C poses a significant burden to children in the region as long as the pandemic continues. MIS-C disproportionately affects black children. The clinical manifestations and outcomes of MIS-C in this region highlight the need for improved surveillance, reporting and data to inform diagnosis and treatment.The online version contains supplementary material available at 10.1186/s12887-022-03308-z. Multisystem inflammatory syndrome in children (MIS-C) emerged in 2020 as a serious paediatric manifestation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been described throughout the world –4. MIS-CWith widespread vaccination in the more-resourced world, it is expected that there will be fewer cases of MIS-C as the pandemic abates. Low and middle-income countries (LMICs) in Africa have the challenge of delayed vaccination, poor vaccine coverage and a relatively larger proportion of younger people in the population . In everChildren under the age of 18 years old admitted to the two main paediatric referral hospitals in Cape Town; the Red Cross War Memorial Children’s Hospital (RXH) or Tygerberg Academic Hospital (TBH) between 7 May 2020 and 31 March 2021 with confirmed MIS-C as per the WHO criteria  were offFrom 17 August 2020 to 31 May 2021 healthy children attending RXH for elective surgery were enrolled with consent and blood was taken and tested for the presence of SARS-CoV-2 antibodies . HealthyNo children in the Western Cape had been vaccinated at the time of recruitment.Race, sex, and comorbidity data were collected for all patients as recorded in the clinical notes. SARS-CoV-2 contact was defined as confirmed (contact with a person with a positive SARS-CoV-2 PCR test in the preceding 6 weeks), suspected (contact with an unwell person with any fever or flu-like symptoms in the preceding 6 weeks) or none. The presence or absence of clinical features over the entire admission as recorded by clinicians was entered directly to an online case report data sheet. These features included fever, rash, diarrhoea, headache, abdominal pain, conjunctivitis, and arthritis.hypotension (defined as blood pressure less than the established range for age recorded > 3 times in 24 h or persistent for more than 1 h)tachycardia (defined as heart rate greater than the established range for age)The presence or absence of the following clinical signs was collected (with definitions):Organ involvement was defined as any documented involvement of either the cardiac, renal, pulmonary, or central nervous systems (CNS). In children who had received an echocardiogram, the minimum ejection fraction (EF) and presence of a pericardial effusion, any valvular disease or coronary artery aneurysms was collected as recorded in the clinical notes by the cardiologist performing the echocardiogram.Maximum: C-reactive protein (CRP), white cell count (WCC), neutrophil count, creatinine, urea, alanine transaminase (ALT), aspartamine transaminase (AST), troponin T (Trop-T), Pro-beta natriuretic protein (BNP), ferritin, D-Dimer, fibrinogen, lactate dehydrogenase (LDH).Minimum: haemoglobin (Hb), sodium, albumin, platelets, lymphocyte count.The following blood test results were collected from each admission:Treatment with antibiotics, intravenous immune globulin (IVIG) and glucocorticoids was recorded. Intensive care unit (ICU) admission, supplementary oxygen therapy, invasive ventilation, inotropic blood pressure support or dialysis treatments and total duration of admission was recorded.In a subset of children from RXH only (41/68), daily data was captured from the notes to estimate the median day of admission after symptom onset, median day of treatment and compare the duration of fever related to treatment. Results from microbiological laboratory tests  were recorded in the 41 patients from RXH only.Children were included after informed consent was obtained and this study was approved by the Human Research Ethics Committee of RXH, TBH and the University of Cape Town .Medians and interquartile ranges were reported due to the nonparametric distribution of these data. Comparison of proportions were measured using Chi squared or Fisher exact test where appropriate and continuous data were summarised using an independent sample t-test or Mann–Whitney U test where appropriate. Analysis of data was performed using SPSS V.27.Sixty-eight children with MIS-C  were recrd wave, so that seroprevalence most likely reflects infection during the first 2 waves .In order to estimate seroprevalence among healthy children during the first 2 waves in the city, 97 healthy children were recruited. The time periods are slightly different for the recruitment of healthy children and those with MIS-C as elective surgeries were cancelled during both waves, so that recruitment of healthy children lagged slightly. Ultimately, all healthy children were recruited prior to the 3p = 0.066) but there was no difference in sex  or ethnicity  by exposure.All healthy children were nasal swab SARS-CoV-2 polymerase chain reaction (PCR) negative and 29/68 (30%) had SARS-CoV-2 antibodies in serum (exposed)  had a confirmed SARS-CoV-2 contact, 18% (11/61) had a suspected contact and 59% (36/61) had no contact reported , tachycardia (98.5%), rash (85.3%), conjunctivitis (77.9%), abdominal pain (60.3%) and hypotension (60.3%) Table . The avep < 0.0001). Eighty five percent of children with MIS-C had gastrointestinal symptoms, including abdominal pain (60.3%), diarrhoea (58.8%) and vomiting (10/41). One child underwent a negative laparotomy for a suspected appendicitis. Nine of 41 children with microbiology lab data (22%) had evidence of other infections during admission. Of these, the majority were judged to be hospital acquired infections or contaminants, but it was determined that two children had a true co-infection in addition to MIS-C, both with evidence of Escherichia coli (E. coli) urinary tract infections (UTI)  of admission (Supp. Table p = 0.001). Children who received IVIG and methylprednisolone vs IVIG alone had no difference in the median maximum CRP , duration of fever (6 days in each group) or duration of hospital stay .Children who received both IVIG and methylprednisolone were more frequently admitted to ICU compared to those that received IVIG alone  required ICU admission, 26 (38.2%) required inotropic blood pressure support, 26 (38.2%) required oxygen therapy, 8 (11.8%) required invasive ventilation and 4 (6%) required peritoneal dialysis. The median hospital stay duration was 7 days . No patients died at either hospital.p = 0.001). Equally, CRP (p = 0.02), troponin T (p = 0.05), Hb (p = 0.05), platelets (p = 0.41) and pro-BNP (p < 0.001) all independently associated with the need for inotropic support. A logistic regression model showed that when controlling for CRP, pro-BNP, Trop T, Hb and platelets, age was a notable predictor of the need for inotropic support .Inotropic support was further explored as a proxy for severity. The median age of children with MIS-C who required inotropic support was higher than those who did not  vs 4 years , The lack of data from Southern Africa may lead to the false conclusion that MIS-C is rare in the region and we hope that these data highlight the burden that African children face from MIS-C, while providing valuable clinical and epidemiological observations in the global discovery of this disease.Cape Town is a relatively well-resourced city in the region, with an established research infrastructure which allows for the rapid and comprehensive documentation of cases of MIS-C in the two main paediatric state hospitals in the city. Although MIS-C was declared a notifiable medical condition in South Africa in September 2020 , the uptThe incidence of MIS-C after infection with SARS-CoV-2 has been difficult to define globally as it has been difficult to accurately estimate the number of children exposed to SARS-CoV-2 in different regions. Reported numbers of SARS-CoV-2 infections in most paediatric populations are inaccurate as children are commonly asymptomatic and may not be tested. In the US, access to testing varied widely between regions over the course of the pandemic, resulting in poor correlation between regional numbers of paediatric SARS-CoV-2 cases and MIS-C cases . Payne eHere, we use the 30% seroprevalence observed in a group of healthy children from the same area and time of exposure as the children with MIS-C, to estimate an incidence of 22 cases per 100 000 SARS-CoV-2 infections in children less than 14 years old in the city. We do not adjust for age or ethnicity, but black children and children under 15 accounted for the highest incidence in the US [We argue that serology gives a more accurate estimation of the denominator - the number of children who have been infected with SARS-CoV-2 - than other studies which most likely explains the lower incidence reported here. This is a small sample of healthy children making the generalisability of the seroprevalence in this sample a weakness. Equally, these children were attending the hospital, which may introduce bias, although this was limited by only recruiting children undergoing elective procedures not associated with underlying inflammatory, immune or other chronic illnesses.As future waves produce multiple infections, it will not be possible to assume that seropositivity associates with a single infection. In the absence of widespread longitudinal community studies which link cases of MIS-C to single (often asymptomatic) infections, we argue that this cohort, in a defined geographic area and time may yield our best  estimation of incidence after infection.This cohort follows the established global trend of MIS-C being more common in Black children. We show no evidence of difference in SARS-CoV-2 exposure in Black children in the healthy cohort, arguing that differences in exposure cannot account for these differences. Although we do not account for many other racial and historical factors relevant to our context, such as access to care, burden of disease and nutrition, we emphasise the possibility that Black children may be at an inherently higher risk of MIS-C and deserve extra vigilance. The demographic distribution of Cape Town is not reflective of the rest of the country where 80% of the population is Black African leading us to believe that there is likely a higher burden of MIS-C throughout the country than is described here, and that efforts to improve monitoring and reporting are urgently warranted.The clinical disease presentation in this cohort is similar to other reported cohorts with rash, abdominal pain, conjunctivitis, tachycardia and hypotension being common. The proportion of children with confirmed exposure to MIS-C is similar to the US (22% vs 24.7%) [Treatment efficacy cannot be inferred from these data, which reflect local treatment recommendations where children who required ICU admission received both glucocorticoids and IVIG. There are no prospective treatment data for MIS-C and retrospective propensity matched analyses are conflicting as to whether IVIG works better alone or in conjunction with glucocorticoids –27. IVIGApproximately one third of cases had any involvement of the brain, kidneys or lungs and nearly half required ICU admission, highlighting the severity of this disease and the requirement for a skilled and multi-disciplinary team. Older age (> 7 years) emerged as an independent predictor of severity (as defined by the requirement for inotropic support), with important clinical utility.In conclusion, we provide a detailed description of a cohort of children with MIS-C in a Southern African setting, with the first seroprevalence based estimation of the incidence of MIS-C after SARS-CoV-2 exposure. These data highlight that MIS-C is a severe disease, causing significant illness in children from this region and most likely others in Africa. We emphasise that the burden of MIS-C in children from this region is under-represented in global data. With the inequality of SARS-CoV-2 vaccination, we expect that MIS-C will persist as a threat to African children. There is an urgent need to improve surveillance and perform research in the region to better inform the diagnosis and management of these children.Additional file 1: Figure S1. Seroprevalence of SARS-CoV-2 in healthy children. 97 children presenting to RXH for elective surgeries that were otherwise well were recruited between August 2020 and May 2021. Twenty-nine of these healthy children had antibodies to SARS-CoV-2  with an estimated seroprevalence of 30% during this period.Additional file 2: Table S1. Pre-existing conditions in children with MIS-C.Additional file 3: Table S2. SARS-CoV-2 exposure.Additional file 4: Table S3. Evidence of infection.Additional file 5: Table S4. Treatment.


V. parahaemolyticus from eastern oysters (Crassostrea virginica). Oysters were inoculated with a 5-strain cocktail of V. parahaemolyticus to levels of 104 to 105 MPN (most probable number)/g and depurated in a controlled re-circulating wet-storage system with artificial seawater (ASW). Both salinity and temperature remarkably affected the efficacy for the depuration of V. parahaemolyticus from oysters during wet-storage. The wet-storage process at salinity 20 ppt at 7.5 °C or 10 °C could achieve a larger than 3 log (MPN/g) reduction of Vibrio at Day 7, which meets the FDA’s requirement as a post-harvest process for V. parahaemolyticus control. At the conditions of 10 °C and 20 ppt, a pre-chilled system could achieve a 3.54 log (MPN/g) reduction of Vibrio in oysters on Day 7. There was no significant difference in the shelf life between inoculated and untreated oysters before the depuration, with a same survival rate (stored in a 4 °C cooler for 15 days) of 93%.This work explored the effects of salinity and temperature on the efficacy of purging  V. parahaemolyticus is a human pathogen that naturally exists in marine environments [Vibrio infections follow a seasonal trend mainly from June to September at high temperatures [Vibrio from growth environments [Vibrio can also replicate in oysters at favorable temperatures [V. parahaemolyticus  can be detected in oysters [V. parahaemolyticus infections were related to eating raw oysters in the U.S. since 1973 [ronments  and primronments . Based oeratures . Oystersronments . Vibrio eratures . Therefo oysters . It is r oysters . It has nce 1973 ,9.V. parahaemolyticus in half-shell Pacific oysters. Ma et al. [V. parahaemolyticus in Pacific oysters. However, both methods lead to the death of the oysters; reducing the Vibrio levels in live oysters is still a challenge. Depuration in UV-sterilized water and electrolyzed oxidizing water was used to solve this problem [Vibrio while keeping oysters alive [V. parahaemolyticus in Pacific oysters (Crassostrea gigas) greater than 3.0 log units [V. parahaemolyticus in eastern oysters (Crassostrea virginica).To increase shellfish safety, the seafood industry employs different strategies intended to reduce or control pathogen levels in oysters . Liu et a et al.  identifi problem ,14. Irrars alive . One preog units . HoweverV. parahaemolyticus from eastern oysters (Crassostrea virginica). We inoculated oysters with V. parahaemolyticus to levels of 104 to 105 MPN/g and depurated them in a controlled re-circulating wet-storage system with artificial seawater (ASW). We explored the effects of salinity, temperature, and the system design on the reduction of the Vibrio, as well as the shelf life of the oysters. In this study, we focused on the reduction of V. parahaemolyticus in eastern oysters and applied a new design of the wet-storage system. We hope those will give new insight into the depuration of Vibrio in seafood.In this work, we investigated the effects of salinity and temperature on the efficacy of purging V. parahaemolyticus levels in oysters was significantly different based on the date of harvest (V. parahaemolyticus levels (<1 log MPN/g), while summer-harvested oysters showed a significant increase in the occurrence of V. parahaemolyticus levels (about 3 log MPN/g). Based on the results, the inoculation procedure can accumulate V. parahaemolyticus levels to ~5 log MPN/g. Most importantly, this inoculation procedure is not dependent on the background level of V. parahaemolyticus in oysters.The occurrence of  harvest . Winter-Vibrio, controlling the temperature at 10 °C . From Day 3, the reduction slowed down to around 0.3 log MPN/g per day. Those results revealed that our wet-storage system had a high deficiency during the first 2 days of the depuration. Depuration of oysters in ASW at 20 ppt resulted in 3.14 log MPN/g of reduction of V. parahaemolyticus in oysters after 7 days, while at 15 and 25 ppt ASW, the reduction only achieved 2.85 and 2.77, respectively.We firstly explored the effect of salinity on the reduction of at 10 °C . In all Vibrio in 20 ppt ASW, which we had already confirmed is the best salinity for depuration. We chose 3 temperatures to test the changes in the V. parahaemolyticus levels during the 7-day wet-storage process  of V. parahaemolyticus at 7.5 °C, the salinity of 20 ppt is shown in We further explored the effect of temperature on the reduction of  process . With thV. parahaemolyticus in inoculated oysters during the pre-chilled (black bar) and non-pre-chilled (grey bar) wet-storage processes. The temperature and salinity of the ASW were controlled at 10 °C and 20 ppt. The reduction of the V. parahaemolyticus levels in a pre-chilled wet-storage process significantly increased to 3.54 log MPN/g. This value is 0.82 log MPN/g higher than a non-pre-chilled depuration system. Furthermore, the pre-chill significantly increased the reduction of the V. parahaemolyticus levels on each day of depuration and the reduction rate.To get better efficiency of depuration for our wet-storage system, we pre-chilled the water tank before the wet-storage process. A total of three studies (100 oysters per study) were conducted to evaluate the survival rate of oysters in the walk-in cooler after the accumulated treatments . All oysVibrio in original oysters for depuration, we scheduled the inoculation method for the accumulation of Vibrio in oysters. Even when we could detect naturally occurring V. parahaemolyticus in oysters harvested in summer, we still needed inoculation to increase the level to specific levels for our wet-storage depuration test. Our results are mostly consistent with findings from current studies [V. parahaemolyticus were detected in all conditions during the 7-day wet-storage test  of wet-storage may favor the depuration of the oysters. However, the behavior of the oysters may be affected when we increase salinity to 25 ppt. Under this condition, the activity of the oysters may be inhibited; hence, Vibrio may not be depurated effectively. Chae et al. [V. parahaemolyticus were observed when oysters were depurated at 15 °C. At low temperatures, the oyster pumps water at the highest level, hence the uptake of Vibrio would be retarded. The temperatures we choose for depuration in our study are all below 15 °C. Based on the results, we found that an optimum wet-storage process could be achieved at a combination of 7.5 °C temperature and 20 ppt salinity, which produced a 3.38 log (MPN/g) reduction after 7 days of depuration . It has been widely applied in many countries to remove sewage-associated bacterial contaminants in different shellfish species sold alive [V. parahaemolyticus in shellfish, depuration has not yet been practiced in the shellfish industry as a post-harvest treatment for controlling the natural flora of shellfish [V. parahaemolyticus in Pacific oysters [V. parahaemolyticus depuration in oysters [V. parahaemolyticus in oysters after 1 day reached up to 2.83. In another study [V. parahaemolyticus in oysters after storage of 1 day at salinities of 20 and 25 ppt, respectively. In a third study [V. parahaemolyticus in oysters also reach up to 2.57 at a salinity of 30 ppt. Those results show better efficiency of depuration than our system. The reasons may be as follows: (1) oyster species—the previous studies used Pacific oysters while we used Eastern oysters for the experiments; (2) original environment—there could be differences in the growing water temperature and salinity ranges for the oysters in different studies, which may affect the activation of oysters during wet-storage; (3) upwelling design of the depuration systems—we notice that downwelling system was applied in previous studies, while in our study, we used an upwelling system. In a downwelling system, the purified water was introduced to the system from the upside and may avoid the resuspension of detritus. This may increase the efficiency of the depuration of the system. Future work will investigate in detail the effects of the design of the systems on depuration. We found many factors that affect the efficiency of wet-storage systems, other than on-site salinity, temperature, etc. In this study, we notice a significant difference in depuration efficiency between pre-chilled and non-pre-chilled tank water for Vibrio in oysters. Furthermore, we assume the source of the oysters (farm-raised or wild-caught) and accumulation method  may affect the reduction of the Vibrio during wet-storage procedures. In addition, the shelf life of the oysters during and after depuration is another concern of the wet-storage process. Further large-scale research is needed to confirm these findings.Depuration has been treated as a post-harvest process for decreasing sewage-associated bacteria  for 15 min. Pelleted cells were resuspended in 30 mL of sterile salt solution (1%) to produce a culture cocktail of approximately 4 × 108 CFU/mL.Pathogenic  and non-pathogenic  is study . Each cuVibrio spp. The ASW was prepared by dissolving Instant Ocean Salt  in deionized water according to the manufacturer’s instructions.Oysters were natural diploid adult eastern oysters sourced from an oyster farm in the Choptank River, MD. Oysters were transported on ice in a cooler to the lab within about 2 h. The oysters, after a brief washing with tap water to remove the mud on the shell, were ready to be inoculated with V. parahaemolyticus in oysters was conducted according to previous studies [V. parahaemolyticus culture cocktail at a level of approximately 104 to 105 CFU/mL. The oysters were held in the tank at room temperature overnight (18 h). The salinity during the inoculation was controlled the same as that in the depuration process. Air was pumped into the water to keep dissolved oxygen (DO) levels favorable for oyster pumping and uptake of Vibrio. Levels of aquaculture oysters were analyzed with a 3-tube most probable-number (MPN) method before and after inoculation.Accumulation of  studies ,17. For V. parahaemolyticus in oysters were analyzed every day for 7 days during the depuration process. A total of three studies (100 oysters per study) were conducted to assess the ability of oysters to survive in the refrigerated walk-in cooler (4 °C) after the accumulated treatments.After inoculation, the oysters were transferred to a laboratory-scale recirculating tank with 150 L of ASW. The system was equipped with an ultraviolet (UV) Lite , a water chiller , a thermometer, and a flow rate meter (1500 L/h). The temperature of the process was set at 7.5, 10, and 12.5 °C, while salinity was fixed at 15, 20, and 25 ppt. The levels of V. parahaemolyticus in oysters were analyzed with a 3-tube MPN method described in the U.S. Food and Drug Administration’s BAM [V. parahaemolyticus isolation and confirmation. The accuracy of using CHROM to determine the MPN values was confirmed by BAX PCR. In a preliminary study, we applied BAX PCR in a few cases to confirm isolates recovered from CHROM agar plates. We did not observe any significant differences between the two methods (using Chrom-agar or BAX PCR to confirm presumptive isolates taken in TCBS plates). Therefore, Chrom-agar was used in all tests of V. parahaemolyticus levels.Levels of on’s BAM , using tV. parahaemolyticus. Then, three different suspicious colonies in each plate were looped to strike on Chromagar plates [For each test, 6 oysters were shucked using a sterile knife on a sterile cutting board and blended in a sterile blender jar with an equal weight of sterile phosphate-buffered saline (PBS) at high speed for 90 s to produce a 1:1 shellfish diluent homogenate. To prepare a 1:10 dilution, 1 g of oyster homogenate (1:1) was mixed with 9 mL of PBS. Additional 10-fold dilutions were prepared using PBS . One-milliliter portions of all dilutions were individually inoculated into 3 tubes of 10 mL Alkaline Peptone Water (APW). APW tubes were incubated at 37 °C for 16–18 h and the positive (turbid) tubes were recorded. A loopful of each positive APW tube was streaked onto Thiosulfate-Citrate Bile Salt-Sucrose (TCBS) plates and incubated at 37 °C for 18–24 h. All plates that had green colonies were considered presumptive positive for r plates  and incup < 0.05. All statistical analyses were based on 3 trials. Samples of each trial were collected from six oysters.Results of microbiological tests were analyzed by using SPSS 19.0 software . Significant differences among means of each treatment over time were established at a level of V. parahaemolyticus in oysters at Day 7. There was no significant difference in the shelf life between inoculated and untreated oysters before the depuration, with the same survival rate (stored in a 4 °C cooler for 15 days) of 93%.The wet-storage process at a combination of 7.5 °C temperature and 20 ppt salinity can achieve a 3.38 log (MPN/g) reduction at Day 7. A pre-chilled system, at the conditions of 10 °C and 20 ppt, can achieve a 3.54 log (MPN/g) reduction of


Furthermore, as a natural compound, it could escape the serious side effects characteristic for approved synthetic PARP inhibitors. Here we present a comprehensive study of toxicological and carcinogenic properties of 7-MG. It was demonstrated that 7-MG does not induce mutations or structural chromosomal abnormalities, and has no blastomogenic activity. A treatment regimen with 7-MG has been established in mice , exerting no adverse effects or changes in morphology. Preliminary data on the 7-MG anticancer activity obtained on transplantable tumor models support our conclusions that 7-MG can become a promising new component of chemotherapy. These Pproteins . Inhibitproteins . We haverticles) . This rerticles) . 7-MG isrticles) . In a rerticles) . From thrticles) .+-binding proteins and nonspecific effects on the organism. Myelodysplastic syndrome/acute myeloid leukemia occurred in some patients after treatment with above-mentioned synthetic inhibitors, and some cases were fatal . These mouse strains are widely used in toxicology studies and in studies involving transplantable tumor models. 7-MG  was administered orally by gavage in experiments A and B ; distilled water and oxoplatin  were used as controls. Adult F1 males and females were examined for the presence of tumors using a binocular microscope.The Ames test was performed in eviously . Three 7eviously . Immortaous work . Five wiThe chromosomal aberration assay was performed using standard procedure . C57BL/62).Six-week-old female CBA mice were obtained from the Stolbovaya farm. Uterine sarcoma US-322 and cervical squamous cell carcinoma RShM-5   were inoThe animal protocols were approved by the Local Committee for Ethics of Animal Experimentation , experiments were conducted in accordance with resolution 81 of the Eurasian Economic Commission and directive 2010/63/EU .t-test , and Fisher’s exact test (study of blastomogenic activity in flies). Significant differences between cells in the comet assay were assessed with the Fisher’s exact test. Data processing was carried out using the Statistica software (StatSoft Inc.).Statistical significance of the difference between animal groups was assessed with the Pearson’s chi-squared test , Student’s 50 values (40–500 mg/kg) predicted for 7-MG with QSAR software ACD/Percepta (www.acdlabs.com). Oral administration of 7-MG to group II , group III , and group IV  was not lethal to any of the animals. Visual observation revealed no apparent lesions or abnormalities of internal organs in treated mice. However, a significant elevation in spleen weight was produced in group IV  is therefore expected to be substantially greater than 600 mg/kg, which allows us to classify 7-MG as only slightly toxic inhibitor.The stimulating effect on lymphoid tissue caused by a single 600 mg/kg dose of 7-MG was then investigated in more detail using different mouse strains: CBA, BALB/c and C57BL/6. Animals were administered with either water or 7-MG (600 mg/kg), and samples of lung, liver, spleen, and intestine were collected after 4 weeks . MicroscThese results lead to a conclusion that 50 mg/kg administration 3 times per week may be an optimal regimen, which is devoid of adverse effects and can be readily applied in further testing for anticancer activity of 7-MG in mice see the .Drosophila melanogaster to assess the mutagenic, recombinogenic and blastomogenic potential)  , comet atential) . The mutitutions . The comitutions , and theitutions . The somnogaster .in vivo, involving various transplantable tumor models and a set of existing drugs as active controls. Below are two illustrative examples demonstrating the utility of 7-MG as a component of chemotherapy.In previous sections, the following findings were made: 1) 7-MG is not carcinogenic and 2) it can be safely administered in an appropriate dosage. As molecular mechanisms of 7-MG are known (PARP1/2 and TGT inhibition), a thorough investigation of its anticancer properties could be initiated Preliminary studies of 7-MG anticancer activity at a safe dose were carried out using mouse transplantable tumor models of uterine sarcoma US-322  and cervPARP1/2 inhibitors represent a novel class of anticancer agents. Although initially proposed for treatment of BRCA-deficient tumors in women (breast or ovarian cancer), these inhibitors have also demonstrated efficacy in other models such as prostate and gastric cancers . Soft tiin vivo. We have established an oral regimen for 7-MG treatment in CBA mice  that exerts no adverse effects or changes in morphology. Adverse events were detected only at a maximum single dose of 600 mg/kg, in the form of small lymphoid infiltrates of non-inflammatory origin in the lungs and liver. These lesions may be resulted from the excessive inhibition of PARP  demonstrated that 7-MG significantly reduces tumor growth at a safe dose and can also potentiate the activity of cisplatin. The interruption of the 7-MG treatment results in accelerated tumor growth, which highlights the advantages of a multiple-dose regimen. The molecular mechanism of 7-MG is fundamentally different from that of drugs like cisplatin. It acts by modulating the enzyme activity instead of causing DNA damage, and the multiple exposure to 7-MG is needed for the effective modulation. The present study of the natural 7-MG compound has confirmed its safety and potential tumor suppressing activity in mice. For further development, it would be important to identify the most sensitive tumors for the 7-MG treatment as well as to select DNA-damaging agents for the combination treatment.